Patterns of activity with periodicities of approximately 24 hours are termed circadian rhythms, and are governed by an internal clock that functions autonomously, but can be entrained by environmental cycles of light or temperature. These behaviors can be entrained to a "zeitgeiber" (most commonly light), but are sustained under conditions of constant darkness and temperature, revealing activity of an endogenous biological clock. Circadian rhythms produced in constant darkness, for example, can also be reset by pulses of light. Such light pulses will shift the phase of the clock in different directions (advance or delay) and to varying degrees in a fashion that depends on the time of light exposure [Pittendrigh, in Handbook of Behavioral Neurobiology, 4, J. Aschoff, Ed., New York: Plenum, 1981, pp. 95-124].
Circadian rhythms appear to be a universal component of animal behavior [Pittendrigh, C. S., Proc. Natl. Acad. Sci USA, 58:1762-1767 (1967); Pittendrigh, C. S., Neurosciences, 437-458 (1974)]. Indeed, circadian physiological rhythms are not limited to the animal kingdom, and genetic screens have identified clock genes in Drosophila [Konopka and Benzer, Proc. Natl. Acad. Sci. USA, 68:2112-2116 (1971); and Sehgal et al., Science, 263:1603-1606 (1994)], Chlamydomonas [Bruce, V. G. Genetics, 70:537-548 (1972)], Neurospora [Feldman and Hoyle, Neurospora crassa Genetics, 75:605-613 (1973); Crosthwaite et al., Science, 276:763-769 (1997)], Cyanobacteria [Kondo et al, Sicience, 266:1233-1236 (1994)], Arabidopsis [Millar et al., Science, 267:1161-1163 (1995)], hamster [Ralph and Menaker, Science, 241:1225-1127(1988)], and mouse [Vitaterna et al., Science, 264:719-725 (1994)].
Fruit flies show circadian regulation of several behaviors [Pittendrigh in The Neurosciences Third Study Program, Chap. 38, F. 0. Schmitt and F. G. Worden, Eds. (MIT Press, Cambridge Mass., 1974); Jackson, in Molecular Genetics of Biological Rhythms, pp. 91-121, M. W. Young, Ed. (Dekker, New York, 1993)]. When populations of Drosophila are entrained to 12 hours of light followed by 12 hours of darkness (LD 12:12), adults emerge from pupae (eclose) rhythmically, with peak eclosion recurring every morning. The eclosion rhythm persists when the entraining cues are removed and behavior is monitored in constant darkness, thus indicating the existence of an endogenous clock. Adult locomotor activity is also controlled by an endogenous clock and recurs rhythmically with a 24-hour period.
In the fruit fly Drosophila melanogaster, two genes are essential components of the circadian clock, period and timeless [Sehgal et al. Science 263:1603 (1994)]. Mutations in either of these genes can produce arrhythmicity or change the period of the rhythm by several hours [Konopka and Benzer Proc. Natl. Acad. Sci. USA. 68:2112 (1971); Sehgal et al Science 263:1603 (1994)]. Molecular studies [Bargiello et al Proc. Natl. Acad. Sci. U.S.A. 81:2142 (1984); Reddy et al Cell 38:701 (1984); Myers Science 270:805 (1995); Hardin et al. Nature 343:536 (1990); Sehgal et al., Science, 270:808-810 (1995)] have shown that per and tim are transcribed with indistinguishable circadian rhythms that are influenced by an interaction of the TIM and PER proteins [Sehgal et al. Science 263:1603 (1994); Gekakiset et al Science, 270:811 (1995)]. A physical association of the two proteins appears to be required for accumulation and nuclear localization of PER [Sehgal et al Science 263:1603 (1994); Gekakiset et al. (1995); Price et al. EMBO J., 14:4044 (1995)]. It is likely that nuclear localization leads to suppression of per and tim transcription [Hardin et al. Nature, 343:536 (1990); Sehgal et al., Science, 270:808-810(1995)]. Cycles of gene expression are thought to be sustained by '5 hour differences in the phases of RNA and protein accumulation. The observed delays in PER accumulation may result, in part, from a requirement for TIM to stabilize PER [Sehgal et al Science 263:1603 (1994); Sehgal et al. Science, 270:808-810(1995); Price et al. EMBO J, 14:4044-4049 (1995)].
More specifically, mutations in the Drosophila period (per) gene, for example, disrupt circadian rhythms of pupal eclosion and adult locomotor behavior [Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971)]. Although per has been cloned and sequenced and its pattern of expression has been analyzed [Baylies et al. in Molecular Genetics of Biological Rhythms, pp. 123-153, M. W. Young, Ed. (Dekker, New York, 1993); Rosbash and Hall Neuron 3:387 (1989)], the biochemical function of the PER protein is unknown. PER shares some homology with a family of transcription factors [Crews et al Cell 52:143 (1988); Nambu et al Cell 67:1157 (1991); Reisz-Porszasz et al Science 256:1193 (1992); Hoffinan et al Cell 252:954 (1991); Burbach et al Proc. NatL. Acad. Sci. U.S.A. 89:8185 (1992)] that possess a common sequence motif called the PAS domain.
Immunocytochemical experiments demonstrated that PER is a nuclear protein in a variety of Drosophila tissues [Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971); Baylies et al in Molecular Genetics of Biological Rhythms, pp. 123-153, M. W. Young, Ed. (Dekker, N.Y., 1993)]. In cells of the adult fly visual and nervous systems, the amount of PER protein fluctuates with a circadian rhythm [Edery et al Proc. Natl. Acad. Sci. U.S.A 91:2260 (1994)], the protein is phosphorylated with a circadian rhythm [Edery et al., Proc. Natl. Acad. Sci. U.S.A 91:2260 (1994)], and PER is observed in nuclei at night but not late in the day [Siwicki et al Neuron 1:141 (1988); Saez and Young Mol. Cell. Biol. 8:5378 (1988); Zerr et al J. Neurosci 10:2749 (1990)]. The expression of per RNA is also cyclic. However, peak mRNA amounts are present late in the day, and the smallest amounts are present late at night [Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971)]. Three mutant alleles--per.sup.O, per.sup.S, and per.sup.L,--cause arrhythmic behavior or shorten or lengthen periods, respectively [Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971)]. These mutations also produce corresponding changes in the rhythms of per RNA and protein amounts [Edery et al Proc. Natl. Acad. Sci. U.S.A 91:2260 (1994); Hardin et al Nature 343:536 (1990); Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992); Sehgal et al Science 263:1603 (1994)] and PER immunoreactivity in nuclei [Sewicki et al Neuron 1:141 (1988); Saez and Young Mol. Cell. Biol. 8:5378 (1988); Zerr et al J. Neurosci. 10:2749 (1990)]. This suggests a possible role for molecular oscillations of per in the establishment of behavioral rhythms [Hardin et al, Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992)].
Several mutations that affect eclosion and locomotor activity have been isolated in behavioral screens [Jackson, in Molecular Genetics of Biological Rhythms, pp. 91-121, M. W. Young, Ed. (Dekker, N.Y., 1993); Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971); Rosbash and Hall Neuron 3:387 (1989); Baylies et al in Molecular Genetics of Biological Rhythms, pp. 123-153, M. W. Young, Ed. (Dekker, N.Y., 1993); Jackson, J. Neurogenet 1:3 (1983); Dushay et al J. Biol. Rhythms 4:1 (1989); Dushay et al Genetics 125:557 (1990); Konopka et al., Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1991)]. The best characterized, and those with the strongest phenotypes, are mutations at the X chromosome-linked period (per) locus [Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971); Rosbash and Hall Neuron 3:387 (1989); Baylies et al in Molecular Genetics of Biological Rhythms, pp. 123-153, M. W. Young, Ed. (Dekker, N.Y., 1993); Jackson, J. Neurogenet 1:3 (1983); Dushay et al J. Biol. Rhythms 4:1 (1989); Dushay et al Genetics 125:557 (1990)]. Missense mutations at per can lengthen or shorten the period of circadian rhythms, whereas null mutations abolish circadian rhythms altogether. The per gene is expressed in many cell types at various stages of development. In most cell types, the period protein (PER) is found in nuclei [James et al EMBO J. 5:2313 (1986); Liu et al Genes Dev. 2:228 (1988); Saez and Young Mol. Cell. Biol. 8, 5378 (1988); Liu et al J. Neurosci. 12:2735 (1992) Siwicki et al Neuron 1:141 (1988); Zerr et al J. Neurosci. 10:2749 (1990); Edery et al Proc. Natl. Acad. Sci. U.S.A. 91:2260 (1994)]. A domain within PER is also found in the Drosophila single-minded protein (SIM) and in subunits of the mammalian aryl hydrocarbon receptor [Crews et al Cell 52:143 (1988); Hoffman et al Science 252:954 (1991); Burbach et al Proc. Natl. Acad. Sci. U.S.A. 89:8185 (1992); Reyes et al Science 256:1193 (1992)], and this domain (PAS, for PER, ARNT, and SIM) mediates dimerization of PER [Huang et al Nature 364:259 (1993)]. The amounts of both PER protein and RNA oscillate with a circadian period, which is affected by the per mutations in the same manner as behavioral rhythms are affected [Siwicki et al Neuron 1:141 (1988); Zerr et al J. Neurosci 10:2749 (1990) Edery et al Proc. Natl. Acad. Sci. U.S.A 91:2260 (1994); Hardin et al Nature 343:536 (1990); Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992)]. Given the homologies to sim and the aryl hydrocarbon receptor (which are thought to regulate transcription), the effects of per on behavioral rhythms have been postulated to depend on circadian regulation of gene expression, including that of per itself [Hardin et al, Nature 343:536 (1990); Hardin et al, Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992)].
Timeless is a second gene which has been associated with circadian rhythms in Drosophila [U.S. patent application Ser. No. 08/619,198 filed Mar. 21, 1996, U.S. Pat. No. 5,885,831, hereby incorporated by reference in its entirety]. In the absence of the Timeless protein, TIM, gene products, such as the Period protein, PER, are not stable in the cytoplasm. Upon binding to the Timeless protein, proteins such as PER are stabilized and translocated into the nucleus. Once in the nucleus, the proteins act to inhibit the production of their own RNA. Both the tim and per genes are transcribed cyclically, and this transcription drives behavior. In particular, the gene products are present in the cytoplasm late in the day when a sleeping cycle is induced, while when the gene products are in the nucleus late at night, and a waking cycle follows.
The TIM protein not only acts as a nuclear translocation factor for the PER protein, but the PER protein also serves as a nuclear translocation factor for the TIM protein, thus indicating that PER and TIM act as mutual and reciprocal nuclear translocation factors. The nuclear translocation of the PER-TIM heterodimer is a crucial step in the regulation of both tim DNA and per DNA transcription.
The TIM protein also plays an important role in entraining the circadian rhythm of Drosophila, and by analogy other animals, to environmental cycles of light. This property of the TIM protein is due to its requirement for stabilizing the PER protein; its role in regulating per DNA transcription; and the TIM protein's extreme sensitivity to light. Unlike the PER protein which requires the TIM protein for stability, the stability of the TIM protein is independent of the PER protein.
Our current understanding of the molecular regulation of circadian rhythmicity in Drosophila comes from integrating genetics and molecular biology. Null mutations in either of two genes, period (per) and timeless (tim), abolish behavioral rhythmicity, while alleles encoding proteins with missense mutations have been recovered at both loci and show either short- or long-period behavioral rhythms [Konopka and Benzer, Proc. Natl. Acad. Sci USA, 68:2112-2116 (1971); Sehgal et al., Science, 263:1603-1606 (1994); Rutila et al, Neuron, 17:921-929 (1996)]. The RNA and protein products of the genes oscillate with a circadian rhythm in wild-type flies. These molecular rhythms are abolished by null mutations of either gene, and the periods of all molecular rhythms are correspondingly altered in each long- and short-period mutant indicating a regulatory interaction between these genes (Hardin et al., Nature, 343:536-540 (1990); Edery et al, Proc. Natl. Acad. Sci USA, 91:2260-2264 (1994); Sehgal et al, Science, 263:1603-1606 (1994); Vosshall et al., Science, 263:1606-1609 (1994); Seghal et al., Science, 270:808-810 (1995); Price et al., EMBO J., 14:4044-4049 (1995); Hunter-Ensor et al., Cell, 84:677-685 (1996); Myers et al., Science, 271:1736-1740 (1996); Zeng et al., Nature, 380:129-135 (1996)].
Production of these molecular cycles appears to depend on the rhythmic formation and nuclear localization of a complex containing the PER and TIM proteins [Seghal et al., Science, 270:808-810 (1995); Gekakis et al., Science, 270:811-815 (1995); Lee et al., Science, 271:1740-1744 (1996); Saez and Young, Neuron, 17:911-920 (1996); Saez and Young, Neuron, 17:911-920 (1996)]. A physical interaction of PER and TIM is required for nuclear localization of either protein, and nuclear activity of these proteins coordinately regulates per and tim transcription through a negative feedback loop [Sehgal et al., Science, 263:1603-1606 (1994); Vosshall et al., Science, 263:1606-1609 (1994); Seghal et al., Science, 270:808-810 (1995); Gekakis et al, Science, 270:811-815 (1995); Hunter-Ensor et al., Cell, 84:677-685 (1996); Lee et al., Science, 271:1740-1744 (1996); Myers et al., Science, 271:1736-1740 (1996); Saez and Young, Neuron, 17:911-920 (1996);. Zheng et al., Nature, 380:129-135 (1996)]. Studies of per.sup.L, a mutation that lengthens the period of behavioral rhythms [Konopka and Benzer, Proc. Natl. Acad. Sci. USA, 68:2112-2116 (1971)] and delays nuclear localization of PER protein [Curtin et al., Neuron, 14:365-372 (1995)], have shown that the PER.sup.L protein has reduced affinity for TIM [Gekakis et al., Science, 270:811-815 (1995). This suggests that rates of PER/TIM association influence the period of the molecular cycle in mutant and wild type flies.
Seghal et al., [Science, 270:808-810 (1995)] proposed a model for the Drosophila clock in which delayed formation of PER/TIM complexes ensures separate phases of per/tim transcription and nuclear function of the encoded proteins. Recent mathematical treatments of the Drosophila data are consistent with this model [Leloup and Goldbeter, J. Biol. Rhythms, 13:70-87 (1998)]. Entrainment of this oscillator is regulated through the TIM protein, which is rapidly eliminated from the nucleus and cytoplasm of pacemaker cells when Drosophila are exposed to daylight [Hunter-Ensor et al., Cell., 84:677-685 (1996); Lee et al., Science, 271:1740-1744 (1996); Myers et al., Science, 271:1736-1740 (1996); Zheng et al., Nature, 380:129-135 (1996)]. Studies of transgenic Drosophila have shown that adult behavioral rhythms can be linked to per and tim expression in a small group of central brain cells, the lateral neurons [Ewer et al., (1992); Frisch et al., Neuron, 12:555-570 (1994); Vosshall and Young, Neuron, 15:345-360 (1995)]. per and tim are also expressed in larval brain cells that are most likely the larval LNs [Kaneko et al., Neurosci., 17:6745-6760 (1997)], suggesting a basis for larval entrainment to light/dark cycles [Sehgal et al., Proc. Natl. acad. Sci. USA, 89:1423-1427 (1992)]. Oscillations of per and tim RNA, and PER and TIM proteins have been found outside of the head in a variety of tissues [Giebultowicz and Hege, Nature, 386:664 (1997); Emery et al., Proc. Natl. Acad. Sci. USA, 94:4092-4096 (1997); Plautz et al., Science, 278:1632-1635 (1997)]. Some of the latter oscillations were observed in vitro with isolated tissues, further indicating a cell autonomous mechanism [Giebultowicz and Hege, Nature, 386:664 (1997); Emery et al., Proc. Natl. Acad. Sci. USA, 94:4092-4096 (1997); Plautz et al., Science, 278:1632-1635 (1997)]. Mammalian homologues of per have recently been identified [Tei et al., Nature, 389 (1997); Sun et al., Cell, 90:1003-1011 (1997); Shigeyoshi et al., Cell, 91:1043-1053 (1997); Albrecht et al., Cell, 91:1055-1064 (1997); Shearman et al., Neuron, 19:1261-1269 (1997)], suggesting that the molecular basis of circadian rhythms may be conserved from flies to mammals. A related circadian oscillator has also been described at the molecular level in Neurospora through the detailed work of Dunlap and colleagues [reviewed by Dunlap et al., Annu. Rev. Genet., 30:579-601 (1996)].
Although key features of the Drosophila clock have been identified, the involvement of additional, essential factors is suspected from prior work. Since neither PER nor TIM has a recognizable DNA-binding motif, an unidentified transcription factor(s) should mediate repression in response to nuclear PER/TIM complexes [(reviewed by Rosbash et al., Harb. Symp. Quant. Biol., 76:265-278 (1996); Young et al., Harb. Symp. Quant. Biol., 61:279-284 (1996)]. PER fails to accumulate in the absence of TIM even in the presence of high per RNA levels [Vosshall et al., Neuron, 15:345-360 (1994); Price et al, EMBO J., 14:4044-4049 (1995)], indicating the existence of an activity that de-stabilizes cytoplasmic PER monomers. Both PER and TIM are phosphorylated with a circadian rhythm [Edery et al., Proc. Natl. Acad. Sci. USA, 94:4092-4096 (1994); Zeng et al., Nature, 380:129-135, 1996)] indicating unidentified kinases. PER, in particular, becomes progressively phosphorylated over many hours, and the timing of this is changed in period-altering mutants, leading to the suggestion that defined hyperphosphorylated form(s) of PER might signal PER degradation [Edery et al., Proc. Natl. Acad. Sci. USA, 94:4092-4096 (1994)].
In summary, circadian rhythms in Drosophila require periodic interaction of the PERIOD (PER) and TIMELESS (TIM) proteins. Physical associations of PER and TIM allow their nuclear translocation, and autoregulation of per and tim transcription through a negative feedback loop. Because PER/TIM heterodimers are only observed when high levels of per and tim RNA have accumulated, self-sustained oscillations are produced in the feedback loop [Gekakis et al., Science, 270:811-815 (1995); Hunter-Ensor et al, Cell, 84:677-685 (1996); Myers et al., Science, 271:1736-1740 (1996); Saez and Young, Neuron, 17:911-920 (1996); Sehgal et al., Science, 270:808-810 (1995) and Zeng et al., Nature, 380:129-135 (1996)]. Although molecular oscillations are maintained in constant darkness for per and tim RNA and for PER and TIM proteins, light can entrain the phases of these rhythms through rapid degradation of the light-sensitive TIM protein [Hunter-Ensor et al., Cell, 84:677-685 (1996); Myers et al., Science, 271:1736-1740 (1996) and Zeng et al., Nature, 380:129-135 (1996)]. Circadian oscillations of PER and TIM phosphorylation have also been described [Edery et al., PNAS, USA, 91:2260-2264 (1994) and Zeng et al., Nature, 380:129-135 (1996)]. However, prior studies have not demonstrated a function for these modifications. The recent identification of several PER homologues from mammals [Albrecht et al. Cell, 91:1055-1064 (1997); Shearman et al., Neuron, 19:1261-1269 (1997); Shigeyoshi et al., Cell, 19:1043-1053 (1997); Sun et al., Cell, 90:10031011 (1997) and Tei et al., Nature, 389:512-516 (1997)] suggests that, like many other biological processes, key molecules and mechanisms involved in circadian rhythms may be evolutionarily conserved between flies and mammals. A related mechanism has also been well defined in Neurospora [cf. Crosthwaite et al., Science, 276:763-769 (1997); Dunlap, Ann. Rev. of Gen, 30:579-601 (1996) and Garceau et al., Cell, 89:469-476 (1997)] and additional genes and proteins are known to play roles in the mouse [Antoch et al., (1997); King et al., Cell, 89:641-653 (1997) and Vitatema et al., Science, 264:719-725 (1994)] the hamster [Ralph and Menaker, Science, 241:1225-1227 (1988)] and Arabidopsis [Millar etaL., Science, 267:1161-1163 (1995)].
Therefore there is a need to identify other factors involved in circadium rythms. Furthermore, there is a need to use such factors to identify agents that can aid in the regulation of biological clocks, including as an aid in overcoming such maladies as jet lag.
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